In the Drosophila visual system, each UV-responsive R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. In a genetic screen based on an R7-dependent behavior, we identified the type I Activin receptor, Baboon, and the nuclear import component Importin-alpha3, as being required for column-specific targeting of R7 axons. In the past year, we have been focusing our study in understanding how Activin-signaling and nuclear import regulate R7 column-specific targeting. [unreadable] Importin-alpha3 or baboon mutant R7s have a novel phenotype: their axons target to the correct layer but extend into neighboring wild-type R7 columns, indicating that the local retinotopic map is defective. Baboon encodes an Activin receptor serine/threonine kinase. In the so-called canonoical pathway, Baboon, in response to Activin, phosphorylates the transcription factor Smad2, which then translocates into the nucleus to regulate transcription. We found that the canonical Activin-signaling components are largely conserved in R7s. Using the in situ hybridization technique, we found that Activin is expressed in R7 and R8, but not in R1-6, neurons during the second target-selection stage. Because Activin mutants were not available, we used dominant-negative and RNA interference approaches to disrupt Activin function in specific tissues. We found that expressing a dominant negative form (DN) or RNAi of Activin in R7s resulted in R7 retinotopy defects that resembled those of baboon mutants. By contrast, expressing either Activin(DN) or Activin RNAi in the medulla neurons using various medulla drivers did not significantly affect R7 connection patterns. Together, these data suggest that Activin functions as an autocrine effector: Activin is both secreted by, and exerts its effects on, R7s. Similarly, removing the downstream transcription factor Smad2 disrupted R7 columnar specificity.[unreadable] Given the similarity of Smad2 and importin-alpha3 mutant phenotypes and their known biological functions, we examined whether these two gene products interact physically. We found that Smad2 co-immunoprecipitated with Importin-alpha3 in vivo, indicating that Smad2 and Importin-alpha3 form a physical complex. To determine the subcellular localization of the Smad2/Importin-alpha3 complexes, we established a primary R-cell culture system and performed immunohistochemistry. We found that the endogenous Importin-alpha3 concentrated in the growth cones as well as in vesicle-like structures along the axons. Interestingly, Smad2 staining largely overlapped with anti-Importin-a3 staining, suggesting that Importin-alpha3 and Smad2 co-localize in axons and growth cones. Because of the known role of Importins in nuclear import, we sought to determine whether nuclear entry of Smad2 depends on importin-alpha3. While Smad2 accumulated in wild-type R7s, in the importin-alpha3 or baboon mutant R7s, Smad2 nuclear accumulation is greatly reduced. Together, these data indicate that Smad2 accumulation in R7 nuclei depends on Importin-alpha3 and Baboon, and that Importin-alpha3 is a component of the Activin-signaling pathway. In addition, the observation that Importin-alpha3 and Smad2 form complexes in axons and growth cones raises the intriguing possibility that Importin-alpha3 plays a role in the retrograde axonal transport of Smad2.[unreadable] Removing Importin-alpha3 or Baboon only causes approximately 20% of the R7 terminals invading their neighbors. The incomplete penetrance of the phenotype suggests the existence of an additional mechanism that functions redundantly to the Activin-signaling pathway. A previous study by Ashley and Katz suggested that competitive/repulsive interactions between adjacent R7s might play a role to restrict R7 terminals to single columns. To determine whether importin-alpha3 and baboon mutant R7 are still subject to repulsion by their neighbors, we tested the effect of removing the R7s adjacent to importin-alpha3 or baboon mutant R7s. To do so, we employed a temperature-sensitive allele of sevenless, v1, which removed most R7s at a non-permissive temperature. We found that wild-type R7 axons form normal synaptic boutons in retinotopically correct columns even in a largely empty R7 terminal field, indicating that disrupting mutual repulsion is not sufficient to affect R7 columnar specificity. By contrast, removing neighboring R7s greatly increased the tendency of importin-alpha3 or baboon mutant R7s to invade adjacent targets. These results suggest that importin-alpha3 and baboon mutant R7s are still responsive to repulsion by neighboring R7s, accounting for their incomplete penetrance of phenotypes. Thus, at least two redundant mechanisms restrict R7 terminals to the correct columns: (i) an extrinsic mechanism mediated by mutual repulsion among R7s; and (ii) an intrinsic mechanism mediated by Activin signaling.